Methods:

Centrifugation:
20 Grams of refrigerated cauliflower was obtained and ground up with
40mL of Mannitol Grinding Buffer and then filtered. 2mL of this solution
was separated as F1 and the remaining was centrifuged (100x gravity
for 30 min. at 4o C). Two distinct layers were created after
centrifugation (pellet 1 or P1 and supernatant 1 or S1). P1 was placed
in ice with 8.0 mL of Mannitol Assay Buffer. 2mL of S1 was stored and
the rest was centrifuged (10,000 x gravity for 30 min. at 4oC). After
centrifugation, S1 formed 2 layers (pellet 2 or P2 and supernatant 2 or
S2). 8.0 mL of Mannitol Assay Buffer was added to P2
Microscopy:
One drop of each fraction (F1, S1, P1, S2, P2) was placed on the slide
with 1 or 2 drops of Azure C dye and observed under the microscope.
Pictures were taken of the observations.
Succinate Dehydrogenase (Part 1):
9 different cuvettes were obtained and each cuvette was filled with
different concentrations of various solutions (Assay Buffer, Azide, DCIP,
Malonate, and Succinate). 3 of the 9 were control cuvettes (Malonate,
Succinate, and Azide). The Spectrophotometer was set to 600 nm
wavelength and each of the cuvettes OD/ absorbance was measured
after the addition of 0.36 mL of the fraction samples. The OD was read
at 0 and 3 minutes intervals of the addition of the fraction sample to
each cuvette.
(Part 2):
10 new cuvettes were obtained and various concentrations of Mannitol
Assay buffer were added to each cuvette (P2, P2A, P2B, P2C, & S2).
Then 0.2 mL of DCIP and 0.2mL of Succinate were added to all the
cuvettes. The Spectrophotometer was set at 600 nm wavelength and
the absorbance values were observed for each of the cuvette after 0
and 3 minutes of adding the fraction samples from P2.
Calculating the Velocity of DCIP Reduction:
First the OD reading of 3 minutes and 0 minutes was found for each
sample fraction (Table 3 & 4). Then the found was divided by DCIP
extinction coefficient (21mM-1cm-1) to find the concentration of
DCIPreduced. Then the amount of the DCIPreduced was found by multiplying
the concentration found above with the volume of reaction. Finally, to
find the velocity, moles of DCIPreduced was divided by the reaction
time.

Results:
Table 3: OD absorbance of
Spectrophotometer
Sample
OD
Fraction
Reading: 0
Mins
Filtrate
0.519
S1

0.839

Sample Fractions through

OD
Reading: 3
mins
1.578

Concentrat
ion
(mM)
1.0086 * 10-4

Velocity (
moles/min
)
3.3619 * 10-

0.994

1.4762 * 10-5 4.9206 * 10-

-2.603*10-6

P1

1.767

1.685

-7.8091 * 10

S2

0.776

0.585

-1.81905*10- -6.063*10-6

P2
Malonate

0.533
0.803

0.544
0.524

1.04762*10-6 3.4921*10-7
-2.65714*10- 5
2.65714*105

Succinate

0.463

0.425

-3.61905*10
6

-1.206*10-6

Azide
0.670
0.731
5.80952*10-6 1.9365*10-6
Table 3: As discussed in methods, various concentrations of
Mannitol Assay Buffer, Azide, DCIP, Malonate, Succinate, and
subcellular fractions were added to the cuvettes (Table 1). The
cuvettes were then placed in a spectrophotometer and the OD
was read at 0 minutes of adding the sample fractions and at 3
minutes.

Table 4: OD absorbance of varied P2 Sample fractions through

spectrophotometer
Sample
OD
OD
Concentrati Velocity (
Fraction
Reading: 0
Reading: 3
ons (mM)
moles/min)
Mins
Mins
P2
0.649
0.782
1.26667*10-5 4.2222*10-6
P2A
0.955
0.996
3.90476*10-6 1.3016*10-6
P2B
0.647
0.730
7.90476*10-6 2.6349*10-6
P2C
0.649
0.676
2.57143*10-6 8.5714*10-7
S2
0.749
0.948
1.89524*10-5 6.3175*10-6
Differing concentrations of Mannitol Assay Buffer, Azide,
Malonate, Succinate, and P2 subcellular fractions were added
to 5 cuvettes (Table 2). The cuvettes were placed in the
spectrophotometer and the OD was read at 0 minutes and then
at 3 minutes after the addition of the sample fractions.
Figure 1. Supernatant 2 (S2) Sample under a microscope

Figure 1: One drop of the sample S2 was dropped on a slide.

Then 2 drops of Azure C were added to the slide. The slide was
then observed under a 40x magnification
Figure 2: Filtrate (F1) sample under a microscope

Figure 2: One drop of the sample F1 was placed on a slide

along with 2 drops of Azure C. The slide was then observed
under the microscope at a 40x magnification

Figure 3. Pellet 1 (P1) Seen under a microscope

Figure 3: 1 drop of P1 was placed on a microscope slide with

Azure C staining. The sample was then observed under a
microscope was a 40x magnification.
Figure 4. Supernatant 1 (S1) Under a microscope

Figure 4: 1 drop of S1 was placed on a microscope followed by

2 drops of azure C staining dye. The sample was observed
under a microscope with a 40x magnification.
Filtrate:
The filtrate solution had a reading of 0.519 at 0 mins and a 1.578
reading at the 3 minutes mark. The reduction velocity of DCIP was
found to be 3.3619 * 10-5 moles/min. Under the microscope, purple
staining was observed (figure 2).
S1:
This specific solution had an absorption of 0.839 at 0 mins and a 0.994
reading at 3 mins. The reduction velocity of DCIP was found to be

4.9206 * 10-5 moles/min. Purple staining was observed under the

microscope (figure 4).
P1 :
The Pellet 1 solution had an OD of 1.767 at 0 mins and a 1.685 reading
at 3 mins. The reduction velocity of DCIP was found to be -2.603 * 10-6
moles/min. Under the microscope, purple staining was observed
(figure 3).
S2 (Table 1):
The supernatant 2, in the spectrophotometer, had the absorption of
0.776 at 0 mins and a 0.585 reading at 3 mins. The DCIP reduction
calculated for S2 was -6.063 * 10-6 moles/min. Though unclear, some
colored solid masses were seen under the microscope (figure 1).
P2 (Table 1):
This specific solution had an absorption of 0.533 at 0 mins and a 0.544
reading at 3 mins. The reduction velocity of DCIP was found to be
3.4921 * 10-7 moles/min.
Malonate Control:
The Malonate solution had an OD of 0.803 at 0 mins and a 0.524
reading at 3 mins. The reduction velocity of DCIP was found to be
-2.657 * 10-5 moles/min.
Succinate Control:
The Succinate Control, in the spectrophotometer, had the absorption of
0.463 at 0 mins and a 0.425 reading at 3 mins. The DCIP reduction
calculated for the control was -1.206 * 10-6 moles/min.
Azide Control:
The filtrate solution had a reading of 0.670 at 0 mins and a 0.731
reading at the 3 minutes mark. The reduction velocity of DCIP was
found to be 1.9365 * 10-6 moles/min
P2 (table 2):
This specific solution had an absorption of 0.649 at 0 mins and a 0.782
reading at 3 mins. The reduction velocity of DCIP was found to be 4.22*
10-6 moles/min (Table 4)
P2A:
The filtrate solution had a reading of 0.955 at 0 mins and a 0.996
reading at the 3 minutes mark. The reduction velocity of DCIP was
found to be 1.3016 * 10-6 moles/min
P2B:

The sample of P2, in the spectrophotometer, had the absorption of

0.647 at 0 mins and a 0.730 reading at 3 mins. The DCIP reduction
calculated for P2B was 2.6349 * 10-6 moles/min.
P2C:
The filtrate solution had an OD reading of 0.649 at 0 mins and a 0.676
reading at the 3 minutes mark. The reduction velocity of DCIP was
found to be 8.5714 * 10-7 moles/min.
S2 (Table 2):
The supernatant 2, in the spectrophotometer, had an absorption of
0.749 at 0 mins and a 0.948 reading at 3 mins. The DCIP reduction
calculated for S2 was 6.3175 * 10-6 moles/min.